JP2002536346A - Method of treating cancer by restoring pp32 function - Google Patents

Abstract

  (57) [Summary] Provided is a method for treating existing cancer or preventing cancer formation by restoring pp32 function in cells lacking normal pp32 activity or expression. The method provides for the recovery of pp32 by either transfecting DNA expressing normal pp32 into cells, administering an inducer of pp32 activation, and / or inhibiting nuclear phosphatase inhibited by pp32.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001] The work leading up to the present invention was in part based on the Grant Number R of the National Institutes of Health.
o1 Supported by CA 54404. The United States government has certain rights in the invention.

BACKGROUND [0002] The present invention relates to various members of a gene family with activity to modulate transformation, and to the technology of mutant-based diagnostics and gene therapy.

Overview of Related Techniques Prostate adenocarcinoma is the most frequent malignancy in adult men, with approximately 317,000 new cases diagnosed each year (Parker et al., CA, 46: 8-27, 1996). Despite the possibility of early diagnosis and treatment (Potosky, et al., JAMA, 273: 548-552, 1995), prostate adenocarcinoma is the second leading cause of death from male cancer after lung cancer.

[0004] To date, studies of mutations in specific genes have had no particular effect on primary prostate cancer. Most mutations in widely studied oncogenes and tumor suppressor genes are associated with overexpression in about 50-60% of those cases.
With the exception of the gene case (Fleming et al., Cancer Res., 46: 1535-1538, 1986), it occurs in only 20-30% of primary prostate cancers. Up to 40% of primary prostate cancers studied by competitive genomic hybridization show chromosomal abnormalities (
Visakorpi et al., Cancer Res., 55: 342-347, 1995), the mutations occurring more frequently upon tumor recurrence and becoming refractory to hormonal therapy. The characteristics of the candidate proto-oncogene or tumor suppressor gene at such a mutated locus may ultimately elucidate the progression of the tumor in the prostate.

[0005] pp32 (GenBank HSU73477) is a highly conserved nuclear phosphoprotein. pp32
Alternatively, increased expression of closely related species is a common feature of clinical cancer.
For example, in human prostate cancer, high levels of expression of RNA that hybridizes to the pp32 probe indicate that clinically significant prostate cancer It occurs in close to 90% (see US Patent No. 5,726,018, incorporated herein by reference).

Molecular characteristics and activity of pp32 pp32 is a differentiation-regulated nuclear phosphoprotein during the differentiation of adult prostate epithelium (Walensky et al., Cancer Res. 53: 4720-4726, 1993). Human pp32 cDNA sequence (G
en-Bank U73477) is 1052 bp long and encodes a 249 amino acid protein. The protein is composed of two domains: an amphipathic amino-terminal α-helical region containing a leucine zipper, and a highly acidic carboxyl-terminal region. The mouse and human pp32 forms are highly conserved, with 90
% Or more nucleic acid homology and 95% or more protein level homology.

[0007] Human pp32 has been uniquely isolated by many groups. Vaesen et al. (“Purifi
cation and characterization of two putative HLA class II associated prot
ein: PHAPI and PHAPII. ”Biol. Chem. Hoppe-Seyler., 375: 113-126, 1994)
Has cloned an essential equivalent molecule named PHPAI from an EBV-transformed human B-lymphoblast cell line; PHAPII cloned with a similar strategy is independent of pp32. In this study, PHAPI was identified by binding to human HLA class II protein in solution and showed membrane and cytoplasmic localization as well as nucleus; the gene was identified by chromosome 15q22.3 by fluorescent in situ hybridization. -q23 (Fink et al., “Localization of the gene encoding the putative hum
an HLA class II-associated protein (PHAPI) to chromosome 15q22.3-q23 by
fluorescence in situ hybridization. "Genomics, 29: 309-310, 1995. More recently, a group studying protein phosphatase inhibitors identified pp32 as an inhibitor of protein phosphatase 2a, I1PP2a (Li et al.). , “
Molecular Identification of I1PP2A, a novel potent heat-stable inhibitor
protein of protein phosphatase 2A. "Biochemistry 35: 6998-7002, 1996)
Another phosphatase inhibitor, I2PP2a, is unrelated to pp32. Interestingly, another recent report (Ulitzur et al., “Biochemical characterization
of mapmodulin, a protein that binds microtubule-associated proteins. ”J
ournal of Biological Chemistry 272: 30577-30582, 1997) identified pp32 as a cytoskeletal-associated cytosolic protein in CHO cells. It is not clear whether this finding stems from system differences or whether pp32 can be located in the cytoplasm under certain circumstances. pp32 has also been identified as a leucine-rich nuclear protein, LANP, in the central nervous system (Matsuoka et al., “A nuclear factor con
taining the leucine-rich repeats expressed in murine cerebellar neurons.
Proc Natl Acad Sci USA 91: 9670-9674, 1994).

[0008] There are also many reports on gene products with a lower degree of homology to pp32. Va
The esen group has developed PHAPI2a (EMBL Locus HSPHAI2A) and PHAPI2b (EMBL Locus
A series of unpublished sequences named HSPHAPI2B) were identified and cloned from an EBV-transformed human B-lymphoblast cell line. These mutant pp32 sequences differ from the sequences reported herein and instead represent the April protein. April cloned from human pancreas is shorter than PHAPI2a by two N-terminal amino acids (Menc
inger et al., “Expression analysis and chromosomal mapping of a novel human
gene, APRIL, encoding an acidic protein rich in leucines. ”Biochmica et
Biophysica Acta. 1395: 176-180, 1998, EMBL Locus HSAPRIL); PHAPI2b
Is identical to the subset of APRIL. Silver-stainable protein SSP29 (unpublished GenB
ank Locus HSU70439) has been cloned from HeLa cells and is identical to PHAPI2a.

[0009] The nuclear phosphoprotein pp32 is associated with proliferation. Malek and colleagues show that various neoplastic cell lines show significantly elevated expression levels, and that bacterial polysaccharides can be expressed by B lymphocytes undergoing polyclonal expansion.
Induced expression of the p32 epitope (Malek et al., J. Biol. Chem.,
265: 13400-13409, 1990). Walensky and colleagues report that pp32 expression levels, measured by in situ hybridization, were based on the Gleason classification of human prostate cancer.
son grade) was reported to increase directly related to the increase.

[0010] Although the pp32 cDNA probe strongly hybridizes to prostate adenocarcinoma, the hybridization signal in normal prostate is restricted to basal cells. Polyclonal anti-pp32 antibody reacts strongly with sections of human prostate adenocarcinoma. The antibodies and riboprobes used by researchers in previous studies were consistent with the reagent cross-reacting with all reported members of the pp32 nuclear phosphoprotein family, and
Although the previous description focused on pp32, it cannot be ruled out that homologous proteins were detected.

SUMMARY OF THE INVENTION [0011] The present invention is directed to altering pp32's tumor suppressor function in cancer by reducing or eliminating expression through control methods that do not involve structural changes to the genome, such as mutations or chromosomal deletions. , Based on the fact that. The subject of the present invention is i) induction of the endogenous pp32 gene by pharmacological or physiological means in cancer cells lacking normal pp32 expression as a form of endogenous gene therapy; or ii) pharmacological or Restoration of pp32 function by physiological means; or, more specifically, iii) restoration of pp32 function by inhibition of pp32-inhibitable nuclear phosphatase by pharmacological or physiological means, leading to the recovery of existing cancers. This is a method of treating cancer by restoring the function of pp32 to prevent treatment or prevent cancer formation.

The present invention is a method of treating certain malignant cells exhibiting less than normal expression of normal tumor suppressive pp32 and / or overexpression of a tumorigenic pp32 mutant, comprising the steps of: A method is provided that includes restoring pp32 functionality. The methods of the invention can be used to treat tumors, neuroendocrine, neuronal, mesenchymal, lymphoid, epithelial or germ cell derived tumors, especially breast and prostate carcinomas.

In a particular embodiment, the method for the treatment of malignant tumor cells of the invention comprises increasing the expression of normal pp32 and / or decreasing the expression of one or more tumorigenic pp32 variants in the cells. Including inducing. Increased expression of normal pp32 can be induced by transfecting cells with DNA that expresses normal pp32, or increased expression of normal pp32 can be induced by administering an inducer of normal pp32 expression be able to. Typically, an inducer of normal pp32 expression will be a small molecule (as opposed to a biological macromolecule) and will affect the regulatory mechanisms for controlling the expression of pp32 and / or its variants Will. The present invention also provides a method of testing a compound to determine whether the compound is an inducer of pp32 expression, comprising the steps of: culturing pp32 and / or
Or measuring the expression of one or more of its variants, herein comprising increasing the expression of normal pp32 in the presence of the compound, or (1
Decreased expression of the tumorigenic pp32 mutant (s) indicates that the compound is an inducer of pp32 expression.

[0014] In another embodiment, the invention provides a method of treating malignant cells that exhibit reduced subnormal expression of normal pp32 and / or overexpression of a pp32 variant, wherein the method comprises: , Protein phosphatases in cells, especially pp32
Inhibiting protein phosphatases that are inhibited by the presence of The protein phosphatase can be a nuclear protein phosphatase, such as a member of the protein phosphatase 2A group. The present invention comprises measuring protein phosphatase activity in cells cultured in the presence and absence of a compound, wherein expression of pp32 in the cells is determined using cells that inhibit protein phosphatase activity, wherein the compound comprises pp32 Also provided is a method of testing a compound to determine if it is an inducer of the function of the compound. Inhibition of protein phosphatase activity by a compound in such a test is an indication that the compound induces pp32 function in cells. The protein phosphatase can be a nuclear protein phosphatase, such as a member of the protein phosphatase 2A group.

[0015] pp32 is a member of a highly conserved family of differentiation-regulated nuclear proteins that are highly expressed in almost all human prostate adenocarcinomas with a Gleason classification of 5 or more. This is in contrast to a low percentage of prostate tumors that express molecular mutations in the proto-oncogene or show tumor suppressor mutations or loss of heterozygosity. Analysis of samples of human prostate adenocarcinoma from three individual patients and paired adjacent normal prostate showed that normal prostate continues to express normal pp32 but prostate adenocarcinoma The three sets of PT-PCR-amplified transcripts from the study demonstrate that all three show multiple cancer-associated coding sequence changes. Cancer-associated sequence changes may be functionally important. Normal pp32 exerts antineoplastic effects through suppression of transformation. In contrast, cancer-associated pp32 mutants increase rather than suppress transformation.

The PC-3 human prostate adenocarcinoma cell line does not express normal pp32. Transfection of PC-3 cell line with normal pp32 results in cell cycle inhibition. The LNCaP human prostate adenocarcinoma cell line expresses normal pp32 and may be defective somewhere in the pathway. LN
Transfection of CaP cell line with normal pp32 does not inhibit cell cycle progression. The expression of pp32 is apparently not deleted through the process of mutation and loss of heterozygosity, but rather uses concomitant induction of expression of tumorigenic members of the pp32 gene family. Down-regulated by other means, this experiment demonstrates that pp32 function can be restored by any method for treating or preventing cancer (see above). Restoring phosphatase inhibition by any means may be sufficient to restore pp32 function, since pp32 may act in whole or in part by inhibiting certain classes of phosphatases.

DETAILED DESCRIPTION OF THE INVENTION The present inventors have discovered that phenotypic alterations in pp32 are a common feature of human prostate cancer. Previous data indicates that 87% of prostate cancers with a Gleason classification of 5 and higher express pp32 or closely related transcripts (US Patent No. 5,734,022, incorporated herein by reference). Incorporated). This is surprising in contrast to the frequency of molecular mutations in other oncogenes and tumor suppressor genes that have been widely studied in primary prostate cancer, which occur in a substantially low percentage of cases. For example, myc overexpression (Fleming et al.) Accounts for approximately 60% of cases.
P53 is abnormal in only about 25% of primary tumors (Isaacs et al., In “
Genetic Alterations in Prostate Cancer. ”Cold Spiring Harbor Symposia o
n Quantitative Biology, 59: 653-659, 1994).

Several lines of evidence suggest that pp32 may act as a tumor suppressor. Functionally, pp32 is ras and myc, mutant p53, Ela in vitro
Or jun or a pair of oncogenes such as human mammary gland virus E6 and E7 (Chen et al., “Structure of pp32, an acidic nuclear
protein which inhibits oncogene-induced formation of transformed foci.
Molecular Biology of the Cell. 7: 2045-2056, 1996. pp32 also inhibits the growth of transformed cells on soft agar (Chen et al.).
Ras-transfected NIH3T3 cells previously transfected to overexpress pp32 do not form cell foci in vitro, or, unlike preliminary cells, do not form tumors in nude mice, unlike control cells . In contrast, in the same system, knockout of endogenous pp32 by an antisense pp32 expression construct
Significantly increases tumorigenicity (Example 12 below).

In clinical prostate cancer, the condition appears initially counterintuitive. Most human prostate cancers appear to express high levels of pp32 by in situ hybridization (see Example 1 below) and staining with heavy anti-pp32 antibody. pp
Since 32 inhibits oncogene-mediated transformation (Chen et al.), Its paradoxical expression in cancer was examined at the sequence level. The paradoxical question of why prostate cancer seems to express high levels of anti-oncogene protein is raised by three patients with normal and adjacent prostate cancer-derived pp32 species and four prostate cancers It was submitted by comparing the sequence and function of pp32 species from cell lines. pp32 is a closely related gene that is expressed in the benign prostate but is located on a different chromosome
pp32r1 and pp32r2 are expressed in prostate cancer. pp32 is a tumor suppressor, but pp32
r1 and pp32r2 are tumorigenic substances. Therefore, pp32, pp32r1, and pp32r2
Alternate use of genes modulates the potential of oncogenes in human prostate cancer. Prostate cancer expresses little or no pp32 and expresses other members of the pp32 family encoded by genes on separate chromosomes. The alternative pp32 gene expressed in prostate cancer is tumorigenic, in sharp contrast to pp32. pp32 is a member of a closely related gene family, and that alternative expression of these closely related genes located on different chromosomes modulates the oncogene potential in human prostate cancer. Shown herein. Mutant pp32 species expressed in prostate cancer are closely related to pp32.

The data of the present invention show that prostate cancer expresses mutant pp32 transcripts, while adjacent normal prostate expresses normal pp32. The two examples clearly show that the expression of alternative genes on different chromosomes can lead to phenotypic switching rather than mutation or alternate splicing. Switching in this molecular phenotype is accompanied by a functional pp32 phenotypic switch. Normal pp32 has characteristically anti-neoplastic characteristics, in contrast to proto-oncogene mutant transcripts that promote oncogene-mediated transformation. This high frequency of abnormalities suggests that expression of the mutant pp32 species may play a pathogenic role in the development of human prostate cancer. In addition, these findings have important implications for diagnosis and prognosis.

Definitions In describing the present invention, the following terms will be used in accordance with the definitions set forth below. Nucleic acids When discussing the structure of a particular double-stranded DNA molecule, the sequence is usually given only in the 5 'to 3' direction along the untranscribed DNA strand (ie, the strand having a sequence homologous to the mRNA). It may be described herein according to convention.

A DNA sequence “corresponds” to an amino acid sequence when the translation of the DNA sequence according to the genetic code results in the amino acid sequence (ie, the DNA sequence “encodes” the amino acid sequence); A sequence "corresponds" to another DNA sequence if the two sequences encode the same amino acid sequence.

[0023] Two DNA sequences are "substantially defined" when at least about 90% (preferably at least about 94%, and most preferably at least about 96%) of the nucleotides match over a defined length of DNA sequence. Same kind. Substantially homologous sequences can be identified by the assay procedures described below, or by isolation and sequencing of DNA molecules. For example, Maniatis et al., Below, DNA Cloning, vols 1 and II, below.
See Nucleic Acid Hybridization below.

A “heterologous” region or domain of a DNA construct is a DNA molecule in a larger DNA molecule in which no relationship to the existing larger molecule can be found.
This is the part where NA can be identified. Thus, when the heterologous region encodes a mammalian gene, the gene will usually be flanked by DNA that is not adjacent to the mammalian genomic DNA in the genome of the original organism. Other examples of heterologous regions are structures in which the coding sequence itself is not found at all (eg, a genomic coding sequence containing cDNA containing introns,
Or a synthetic sequence with codons different from the original gene). Allelic
) The case of alteration or spontaneous mutation is defined as heterologous as defined herein.
Does not give rise to DNA regions.

A “coding sequence” or “open reading frame” is an in-frame sequence of codons corresponding to or encoding a protein or peptide sequence (in view of the genetic code). Two coding sequences correspond to each other when their sequences or their complementary sequences encode the same amino acid sequence. Coding sequences associated with appropriate regulatory sequences can be transcribed and translated into polypeptides in vivo. The polyadenylation signal and transcription termination sequence will usually be located 3 ′ to the coding sequence. A “promoter sequence” is capable of binding RNA polymerase in a cell and initiating transcription of a downstream (3 ′ direction) coding sequence
It is a DNA regulatory region. A promoter sequence typically includes additional sites for the attachment of regulatory molecules (eg, transcription factors) that affect the transcription of the coding sequence. Is the coding sequence "under control" of the promoter sequence when RNA polymerase binds to the promoter sequence in the cell, transcribes the coding sequence into mRNA, and is then translated into the protein encoded by the coding sequence. Or "operably linked" to a promoter.

A vector is used for introducing a foreign substance such as DNA, RNA or protein into an organism. Typical vectors include recombinant viruses (for DNA) and liposomes (for proteins). A "DNA vector" is a replicon, such as a plasmid, phage, or cosmid, to which another DNA portion may be attached, thereby causing replication of the attached portion. An "expression vector" is a DNA vector that contains control sequences that will direct protein synthesis by a suitable host cell. This usually refers to a promoter for binding RNA polymerase to initiate transcription of the mRNA, as well as a ribosome binding site and initiation signal to direct translation of the mRNA into a polypeptide. Following incorporation of the DNA sequence into the expression vector at the appropriate site and in the correct reading frame, transformation of the appropriate host cell with the vector results in the DN.
Enables production of the protein encoded by the A sequence.

The expression vector may alternatively contain an antisense sequence, in which a small DNA fragment corresponding to all or part of the mRNA sequence is inserted into the vector in the opposite direction after the promoter. . As a result, the inserted DNA is transcribed to be complementary and m
It will produce RNA that can bind or hybridize to the RNA. Upon binding to the mRNA, translation of the mRNA is hampered and consequently the protein encoded by the mRNA is not produced. The production and use of antisense expression vectors is described in US Pat. No. 5,107,065 (which describes and illustrates antisense control of genes in plants) and US Pat. , Exemplifying prokaryotes), both of which are incorporated herein by reference.

“Amplification” of a nucleic acid sequence is the production of multiple copies of a particular nucleic acid sequence in vitro. Amplification sequences usually take the form of DNA. Various techniques for performing such amplifications are described in review articles by Van Brunt (1990, Bio / Technol., 8 (4): 291-294). Polymerase chain reaction or PCR is the basic type of nucleic acid amplification,
The use of PCR herein should be considered as representative of other suitable amplification techniques.

Polypeptides For purposes of clarifying the present invention, two proteins are homologous if 80% of the amino acids in each amino acid sequence are the same; for proteins of different lengths, the sequences are common. At least 80% will be the same over the same sequence (ie, shorter protein length).

[0030] The two amino acid sequences have at least about a defined length of the amino acid sequence.
"Substantially homologous" when 87% of the amino acids are matched, preferably at least about 89%, more preferably at least about 95%. Typically, two amino acid sequences of the same type will differ only by conservative substitutions.

A “conservative amino acid substitution” is the replacement of one amino acid residue in a sequence by another amino acid residue of the same property, resulting in substantially the same secondary and tertiary structure of the peptide. Becomes Conservative amino acid substitutions occur when an amino acid has substantially the same charge or hydrophobicity as the amino acid it replaces, and the substitution has no significant effect on the local structure of the protein. Sets of amino acids that can make conservative substitutions for one another are well known to those skilled in the art.

The polypeptides of the present invention include pp32r1 and pp32r1 analogs, pp32r2 and pp32r2 analogs, and other variants of pp32 and their analogs. pp32r1 and pp
32r2 is a naturally occurring, mature protein, and furthermore, pp32r1 and
Includes all precursors and allylic variants of pp32r2, as well as different molecular weight forms that may result from inconsistent processes in vivo. An example of the pp32r1 sequence is shown in the upper row of FIG. “Pp32r1 analogs” are: 1) “allelic variants of pp32r1”, which are polypeptides substantially homologous to pp32r1. Preferably, the amino acid sequence of the allelic variant is encoded by a nucleic acid sequence in which one nucleotide out of 300 nucleotides differs from the sequence of pp32r1; 2) a "truncated pp32r1 peptide", which is preferably 3) "pp32r1", including fragments of either pp32 or an allelic variant of pp32r1 possessing either (ii) an amino acid sequence unique to pp32r1, (ii) an epitope unique to pp32r1 or (iii) pp32r1 activity; Fusion protein ", which is the above polypeptide (pp32r1, an allelic variant of pp32r1 or a truncated pp32r1) fused to any of the heterologous amino acid sequences
A heterologous polypeptide composed of one of the above peptides.

[0033] The "unique" sequence, amino acid sequence or the nucleic acid sequence encoding them, of a pp32r1 variant according to the invention is identical to the sequence of the pp32r1 polypeptide, but is human pp32 (Genbank Locus HSU73477) , Mouse pp32 (Genbank Locus MMU73478
), Human cerebellar leucine-rich acidic nucleoprotein (LANP) (Genbank Locus AF0256)
84), mouse LANP (Genbank Locus AF022957), I1PP2a or human potent thermostable protein phosphatase 2a inhibitor (Genbank Locus HSU60823), SSP29 (G
enbank Locus HSU70439), HLA-DR related protein I (Genbank Locus HSPPHAPI,
Deposit number X75090), PHAPI2a (EMBL Locus HSPHAPI2A, Genbank deposit number Y07569)
, PHAPI2b (EMBL Locus HSPHAPI2B, Genbank accession number Y07570), and April (E
At least one amino acid or nucleotide residue differs from that of MBL Locus HSAPRIL), and is preferably a sequence not found anywhere in the human genome (a list of these sequences is provided in Table 3A). Similarly, the epitope is pp32
"Unique" to a pp32rl polypeptide when found in the r1 polypeptide but not in any member of the set of proteins listed above. Analogs of pp32r2 and unique pp32r2 sequences are similarly defined. Of course, the unique sequence of pp32r1 is not found in pp32r2 and vice versa.

“Variants of pp32” are homologous proteins that differ in at least two amino acids from pp32. In particular, a sequence comparison between pp32 and the variant will reveal at least one 10 amino acid portion that differs by at least 2 (2) amino acids in the sequence. More typically, a variant will exhibit at least two of its ten amino acid portions. Preferably, a variant of pp32 according to the invention has a functional activity of pp32
Would be different. In particular, pp32r1 and pp32r2 are variants of pp32 whose activity includes stimulation of transformation in the rat fibroblast transformation assay described herein.

In a composition comprising selected Component A, when Component A comprises at least about 75% by weight of the combined weight of Components A and B, another Component B is “substantially free (fre
e) ". Preferably, the selected component A comprises at least about 90% by weight of the mixture weight, most preferably at least about 99% by weight of the mixture weight. In the case of compositions that contain and are substantially free of contaminating proteins, it can be noted that a composition having the activity of the protein of interest contains a species having only a single molecular weight (ie, a "homologous" composition). Is preferred.

As used herein, “biological sample” refers to a sample of tissue or fluid isolated from an individual, such as plasma, serum, spinal fluid, lymph, skin, Samples of respiratory, intestinal, and urogenital tract outer sections, tears, saliva, milk, blood cells, tumors, organs, and cell culture constructs in vivo (cells in cell culture medium, putative virus-infected cells , Recombinant cells, and conditioned media that cause the growth of cellular components, including, but not limited to).

“Human tissue” is a clump of human cells that can constitute a solid mass. The term also includes suspensions of blood cells or human cells, such as human cell lines. The term "immunoglobulin molecule" includes whole antibodies composed of four immunoglobulin peptide chains, two heavy chains and two light chains, as well as immunoglobulin fragments. “Immunoglobulin fragments” are protein molecules associated with an antibody that are known to have binding specificity for an epitope of the original antibody, such as Fab, F (ab) ′ ₂ , Fv. Two polypeptides are "immunologically cross-reactive" when both polypeptides react with the same monoclonal antiserum.

General Methods The practice of the present invention will employ, unless otherwise indicated, conventional molecular biology, microbiology, and recombinant DNA techniques within the art. Such techniques are well known to those skilled in the art and are fully explained in the literature. For example, Maniatis, Fritsch & Sambro
ok, "Molecular Cloning: A Laboratory Manual"(1982);"DNA Cloning: A Pra
ctical Approach, "Volumes I and II (DN Glover, ed., 1985);" Oligonucle
otide Synthesis "(MJ Gait, ed., 1984);" Nucleic Acid Hybridization "(B
.D. Hames & SJ Higgins, ed., 1985); "Transcription and Translation" (B
.D. Hames & SJ Higgins, ed., 1984); "Animal Cell Culture" (RI Freshn
ey, ed., 1986); "Immobilized Cells and Enzymes" (IRL Press, 1986); B. Pe
rbal, "A Practical Guide to Molecular Cloning" (1984), and Sambrook et al.,
See "Molecular Cloning: a Laboratory Manual" (1989).

Screening of the human genomic library in bacteriophage using probes generated from pp32-related genomic DNA human pp32 cDNA produced new sequences containing an open reading frame encoding a protein homologous to pp32. Example 2
Pp32r2 sequence reported in Chen et al., Mol. Biol. Cell, 7: 2045-2056, 1996). The pp32r1 and pp32r2 sequences (see FIGS. 2 and 5) are substantially homologous to pp32, but due to multiple single nucleotide base changes and short deletions, they may be encoded by genes different from the pp32 gene It is suggested. The pp32 family is
Other reported substantially homologous polypeptides: HLA-DR related protein I (Vaesen,
1994), leucine-rich acidic nucleoprotein (Matsuoka, 1994), and protein phosphatase 2A inhibitor (Li, 1996).

[0040] A DNA portion or oligonucleotide having a specific sequence can be chemically synthesized or isolated by one of several approaches. Desired DN
The basic strategies for identifying, amplifying and isolating A sequences and assembling them into larger DNA molecules containing the desired sequence domains in the desired order are well known to those skilled in the art. See, for example, Sambrook et al., (1989); B. Perbal,
(1984). Preferably, the DNA portions corresponding to all or part of the cDNA or genomic sequence of pp32r1 can be individually isolated using the polymerase chain reaction (MAInnis et al., “PCR protocols: A Guide To Methods and Applications,”
Academic Press, 1990). Complete sequences can be assembled by overlapping oligonucleotides prepared by standard methods, and can be assembled into a complete coding sequence. For example, Edge (1981) Nature 292: 756; Nambair
(1984) Science 223: 1299; Jay et al. (1984) J. Bio. Chem., 259: 6311.

The assembled sequence can be cloned into a suitable vector or replicon, where it can be maintained as a composition substantially free of vectors that do not contain the assembled sequence. This provides a reservoir of the assembled sequence, and partial or total sequences can be extracted from the reservoir by cutting out DNA in the reservoir material using restriction enzymes or by PCR amplification. Many cloning vectors are known to those of skill in the art, and the selection of an appropriate cloning vector is a matter of choice (see, eg, Sambrook et al., Which is incorporated herein by reference). ). Construction of a vector containing the desired portions of the DNA linked by the appropriate DNA sequence is accomplished by techniques similar to those used to construct the portions. These vectors can be constructed to contain additional DNA portions, such as a bacterial origin of replication to create a shuttle vector (for shuttles between prokaryotic and mammalian hosts).

Procedures for constructing or expressing proteins of defined sequence are well known in the art. The DNA sequence encoding pp32r1, pp32r2, or an analog of either pp32r1 or pp32r2 contains primer extension, linker insertion and P
It can be chemically synthesized or prepared from wild-type sequences by one of several approaches, including CR (see, eg, Sambrook et al.). Mutants having additions, deletions, and substitutions in the wild-type sequence can be prepared by these techniques.
It is preferred that the variants be tested to ensure that they are the desired sequence by sequence analysis and / or the assays described below. The mutant protein to be tested can be prepared by placing the coding sequence of the polypeptide in a vector under the control of a promoter, such that the DNA sequence is transformed in a host cell transformed with the (expression) vector. It is transcribed into RNA and translated into protein. Variant proteins can be produced by growing a host cell that is transfected with an expression vector containing the variant coding sequence under conditions in which the polypeptide is expressed. Selection of appropriate growth conditions is within the skill of the art.

The assembled sequence can be cloned in either a suitable vector or a replicon, where it can be maintained as a composition substantially free of vectors that do not contain the assembled sequence. This provides a reservoir of the assembled sequence, and partial or total sequences can be extracted from the reservoir by cutting out DNA from the reservoir material using restriction enzymes or by PCR amplification. Many cloning vectors are known to those of skill in the art, and the selection of an appropriate cloning vector is a matter of choice (see, eg, Sambrook et al., Which is incorporated herein by reference). ). Construction of a vector containing the desired portions of the DNA linked by the appropriate DNA sequence is accomplished by techniques similar to those used to construct the portions. These vectors can be constructed to contain additional DNA portions, such as a bacterial origin of replication for constructing shuttle vectors (for shuttle between prokaryotic and mammalian hosts).

Production of Recombinant Peptides Preferably, DNA from the selected clone should be subcloned in an expression vector, and the proteins expressed by cells transformed with the vector are described below. Should be tested for immunoreactivity using an antibody against the recombinant protein of the invention prepared as such. Such subcloning is readily within the skill of one in the art in light of the present disclosure. The DN of the present invention
The amino acid coding region of the A sequence is a recombinant peptide expressed by the DNA sequence, pp32.
r1, pp32r2, or other desired pp32 variants as long as they retain at least one epitope that cross-reacts with an antibody that specifically immunoreacts with the variant pp32, or longer or shorter than the coding region of the disclosed sequences. May be. The preparation of selected clones containing DNA sequences corresponding to all or part of the sequence of pp32r1 or pp32r2 can be accomplished using conventional molecular biology techniques in addition to the information provided herein. Can be accomplished by a vendor.

From a suitable tissue / fluid source, pp such as pp32r1 that cross-reacts with antibodies specific for pp32
It is possible to purify 32 variant proteins; however, cross-reactive pp32 variants, or analogs thereof, can also be produced recombinantly from DNA sequences encoding such proteins or polypeptides. Polypeptides corresponding to the recombinant proteins of the present invention can be expressed in cells using an expression vector containing DNA from a clone selected from a mammalian (preferably human) library, as described herein. It can be obtained by transformation. Suitable expression vectors and host cell systems are well known to those of skill in the art and are taught, for example, in Sambrook et al., 1989. Peptides can be obtained by growing transformed cells cultured under conditions that allow expression of the cloned DNA. Of course, the peptide expressed by the clone may be pp32r1 or pp32r1 as long as the peptide immunologically cross-reacts.
It may be longer or shorter than pp32r2. Depending on the expression vector selected, the peptide may be secreted or retained in a cell, a fusion protein,
Or as a mature protein or as an encapsulated protein (
Inclusion protein). The desired polypeptide, with or without cell disruption, can be recovered from the culture by well-known procedures such as centrifugation, filtration, and extraction, depending on the mode of peptide expression. The crude aqueous solution or suspension can be enriched for the desired peptide by protein purification techniques well known to those skilled in the art. Preparation of the polypeptide is performed by post-cultivation
bioprocessing of proteins containing foreign sequences that can be removed by reprocessing.

Using the nucleotide sequences disclosed herein and the polypeptides expressed therefrom, one has a high binding affinity for pp32r1 or pp32r2, but other variants of pp32 and / or pp32 Antibodies with considerably lower affinity can be obtained. Such antibodies, whether monoclonal or purified polyclonal, can be used to specifically detect pp32r1 or pp32r2. Diagnostic assays suitable for the detection of pp32, as well as techniques for preparing polypeptides, antibodies and nucleic acid probes for use in diagnostic procedures, are described in U.S. Patent Nos. And these techniques can be applied to pp32r1 or pp32r2 by substitution of the nucleic acid sequences disclosed herein. Similar substitutions may be made for other variants of pp32.

Pp32r1 Promoter Sequences Consensus sequences that bind to activated steroid receptors found in genomic sequences upstream from the pp32r1 coding region are consistent with hormonal control of gene expression. The consensus sequence has been implicated in both the induction and repression of expression by steroid hormones. Due to the combination of both positive and negative acting elements, pp32
Complex regulation of r1 expression is suggested.

Possible steroid hormone regulation of pp32r1 expression is important for prostate cancer. Approximately half of the treated patients respond initially to androgen ablation, but subsequent hormonal refraction and continued aggressive tumor growth are common (Garnick, M.
.B., “Prostate Cancer,” in Scientific American Medicine, Dale, DC an
d Federman, DD Eds., Scientific American Inc., New York, 1995). Many different steroid hormones control prostate cancer cell growth (Huggins et al., “S
tudies on prostate cancer: I. The effect of castration, of estrogen, and
of androgen injection on serum phosphatases in metastatic carcinoma of
the prostate, "Cancer Res., I: 293, 1941). These findings have established the basis for androgen ablation therapy for the treatment of metastatic prostate cancer.

The present invention provides an androgen-activated promoter based on the upstream portion of the genomic sequence of FIG. The promoter sequence provided by the present invention binds to the 3 'end of the coding sequence by the translation start codon and extends upstream (5' direction) to initiate transcription at least at levels above background. Contains the required number of bases or elements. Within the promoter sequence is a transcription initiation site (conveniently defined by mapping with nuclease S1), a protein binding domain (consensus sequence) (TATA consensus sequence) located approximately 100 bases upstream of the transcription initiation site, generally the TATA box. Box binding proteins and binding sites for RNA polymerase),
And upstream of the TATA box, various other protein binding domains (consensus sequences) will be found that modulate the transcription initiation site and the basic transcriptional activity of the TATA box. Preferably, various other protein binding domains include (1) androgen receptor, estrogen receptor, glucocorticoid receptor, and progesterone receptor; (2) Fos, Jun, JunB, and Myc. Tables for binding transcription factors containing, but not limited to, leucine zipper motifs; and (3) specific tissue-specific transcription factors, including but not limited to GATA-1 and GATA-2. Contains the recognition sequence shown in 1. TATA
Various other protein binding domains upstream of the box are responsible for promoter specificity (
Tissue-specific expression), accuracy (correct onset), and strength (transcription frequency). Promoter elements provide overlapping functions, such that a promoter can function in the absence of a subset of these elements.

Therapy Inhibiting the ability of any of the pp32 variants to protransform by any means would be expected as a way of achieving therapy. Phenotypic changes in pp32 due to the use of alternative genes are a common feature of human prostate cancer, and changes in pp32 phenotype are associated with changes in oncogene potential. Modulation of the oncogene potential by the use of alternative genes has interesting implications.
Preliminary comparisons of the structures predicted by the energy minimization program for pp32 and mutant pp32 species show that critical structural A difference is suggested. From a temporal point of view, it is not yet clear to what extent the use of alternative pp32 genes of the pp32 family occurs early in the neoplastic process. Events common to the use of the alternative pp32 gene in prostate cancer suggest that it may be an early important event in tumorigenesis.

[0051] Surrogate gene use is potentially reversible and has additional important clinical and mechanical implications. The malignant potential of tumor cells has been identified in the form of endogenous gene therapy
It can be modified by manipulating the expression pattern of members of the pp32 family. Several possibilities exist. One possibility is that mutations in pp32 located at 15q22.3-q23 and loss of heterozygosity (LOH) somehow induce increased expression of pp32r1 and pp32r2. However, LOH is unlikely because the chromosomal deletion at 15q22.3-q23 is not a common feature of prostate cancer. Even more interesting from both a pathogenic and therapeutic point of view is that they may involve abnormal forms of control. Epigenetic regulation may induce inactivation of the pp32 gene and concomitant activation of the pp32r1 and pp32r2 genes by means such as methylation and demethylation. Another mechanism is that while inducing pp32r1 and pp32r2, the differential
It may involve regulation by one or more transcription factors that repress 32.
Eventually, post-transcriptional mechanisms may also be elicited, whereby differential expression will be controlled by changes in mRNA or protein stability. From a carcinogenesis point of view, all of the latter mechanisms will contribute to tumorigenesis through plastic and potentially reversible changes in control over irreversible structural changes in the genome. Thus, it is envisioned that the pp32 family will ultimately be used as a target for pharmacological prophylactic and therapeutic strategies in prostate cancer.

US Pat. No. 5,726,018, which is hereby incorporated by reference, describes various therapeutic methods that can be applied to those skilled in the art based on the nucleotide and protein sequences disclosed herein. . In particular embodiments, all or part of the sequence of pp32r1 or pp32r2 is provided in an antisense orientation and may inhibit expression of a variant found in cancer, particularly prostate cancer. Suitable methods for preparing antisense expression vectors and administering antisense therapy can be found in US Pat. No. 5,756,676, incorporated herein by reference. It is preferable to prescreen a patient population using the diagnostic methods described herein to identify patients with tumors that express a particular pp32 variant.

Screening for compounds that have a therapeutic effect on prostate cancer may also be facilitated by the present invention. A study that can be used to screen candidate compounds is described in U.S. Patent No. (See example below). Compounds that affect steroid-dependent protein expression are also functionally coupled to DNA sequences capable of detecting their expression, and similar screening studies using the androgen-activated promoter provided herein. Can be detected according to the present invention. (Marker sequences are well known in the art; see, for example, Sambrook et al., The selection of an appropriate detectable expression marker is routine for one of ordinary skill in the art.) Screening by testing the effect of the candidate compound on recombinant cells containing an expression vector having an androgen-activated promoter that functionally couples with an expression marker can be performed using the promoter sequences provided herein. Consideration is within the skill of the art. In one aspect, the invention provides (1) cells transfected with a DNA molecule containing an androgen-activated transcription promoter that is operably linked to an open reading frame comprising at least one exon of a protein coding sequence. Methods for screening candidate compounds for pharmacological activity by culturing and (2) determining expression of the open reading frame in the presence and absence of the compound. In a preferred embodiment, the androgen activating promoter may be part of the sequence in FIG. 2 upstream of the translation start site, or the androgen activating promoter may be 2700 bp upstream of the translation start site. Similar experiments for screening potential therapeutic compounds are described in International Patent Publication (International
l Patent Publication) According to the screening method described in WO 99/29906, Jun 17, 1999, it can be carried out using a cell line expressing a pp32 mutant such as pp32r1 or pp32r2.

Diagnostic Methods Based on the pp32 Gene Family In one aspect, the invention provides methods for detecting and identifying members of the pp32 gene family. As described herein, the presence of one or more members of a gene family is detected using an assay based on common structure among members resulting from common or similar sequences. Can be done. For example, polyclonal antibodies induced by pp32 will cross-react with pp32r1 and pp32r2, which contain various alleles of these pp32 variants. Similarly, the complete coding region of the pp32 cDNA contains the mutant variant Will hybridize under suitable conditions with a nucleic acid encoding any of the following (Example 1). The choice of conditions that promote immune cross-reactivity or cross-hybridization required for such detection is within the skill of the art in view of the examples provided herein. For example, by using a large nucleotide probe in a hybridization experiment, the effect of one or several differences in sequence can be overcome, i.e., a larger probe will bind to a less similar target sequence. In contrast, shorter probes will contribute a greater percentage of each nucleotide to affinity, and mutation of one nucleotide will cause a greater relative decrease in hybridization efficiency. A typical probe of 50 or more nucleotides was used to find homologues to a given sequence, and the studies reported in Example 1 used the entire sequence of pp32 as a probe except for pp32. Cells that express homologous members of the gene family were found. Similarly, polyclonal antibodies that elicit against antibodies with multiple epitopes are more likely to cross-react with secondary antigens having several identical epitopes plus several identical epitopes, while monoclonal antibodies Even antibodies or even purified polyclonal antisera may not bind to secondary antigens.

In addition to determining the presence of one or more members of the pp32 gene family, the present invention also provides a method for discriminating between members. For example, determine which pp32 variants may be useful to determine whether a pro- or suppressive variant is present in a tissue sample. Suitable methods for identification include both immunoassays and nucleic acid binding assays. Target analyte (
detection ligand (eg, antibody or oligonucleotide) for the analyte)
A method capable of detecting a 10-fold difference in affinity of is preferred. Such methods have been well described in other systems and, by adopting them, given the guidance provided herein, routine modifications of such methods allow for the exchange between pp32 mutants. Can be identified.

Assays at the protein level rely on monoclonal or purified polyclonal antibodies of relatively high affinity for one variant as compared to another (eg, Smith et al. (Changes governing antibody-hapten interaction). The main kinetics that occur is the dissociation rate of the complex, indicating that the strength of the antibody-hapten relationship is essentially determined by the activation energy of dissociation, “Kinetics in interact.
ions between antibodies and haptens, ”Biochemistry, 14 (7): 1496-1502, 19
75), and Pontarotti et al. (A set of monoclonal antibodies capable of binding different dimeric alkaloids and discriminating alkaloid haptens due to the different relative affinities of the various monoclonal antibodies to specific dimeric alkaloids. To describe,
“Monoclonal antibodies to antitumor Vinca alkaloids: thermodynamics and
kinetics, "Molecular Immunology, 22 (3): 277-84, 1985), each of which is incorporated herein by reference.) The relative affinities of monoclonal antibodies with different affinities Modifications suitable for the conditions of the immunoassay for highlighting are also discussed in U.S. Patent No. 5,759,791, which is incorporated herein by reference.

Many methods are available that allow discrimination between nucleic acid sequences that differ by as little as one nucleotide in the sequence. For example, ligase chain reaction has been used to detect point mutations in various genes (see, eg, Abravaya et al., “Detection of point m
utations with a modified ligase chain reaction (Gap-LCR), ”Nucleic Acid
s Research, 23 (4): 675-82, 1995, or Pfeffer et al., “A ligase chain rea.
ction targeting two adjacent nucleotides allows the differentiation of c
owpox virus from other Orthopoxvirus species, ”Journal of Virological M
ethods, 49 (3): 353-60, 1994, each of which is incorporated herein by reference). Sequence amplification by PCR may also be used to synthesize suitable primers, for example, short primers, preferably 10-15 matched nucleotides (where at least one of the primers matches one variant but is compatible with another variant). Have a unique base at the 3 'end that does not match) and that primers with unique bases have at least a 10-fold reduction in the off-rate between perfectly matched and non-matched variants. Sequences can be identified by using annealing conditions with a fold difference. Similar discrimination can be achieved with short probes (typically less than 50 bp, preferably 2 bp).
5 bp or less, more preferably 20 bp or less or even 10-12 bp)
Adjusting the conditions of the hybridization reaction such that at least a 10-fold difference in off-rate is achieved for the probe between perfectly matched and non-matched variants, Can be achieved in other methods depending on hybridization. Cleavage enzyme fragment length polymorphisms can also be used, and the specific examples below show that, given the sequences provided herein, those skilled in the art Provide guidance that can be set.

The diagnostic methods of the present invention can be used for prophylactic purposes and for patient identification as described herein. In particular, the method of the present invention allows for the identification of products expressed from the various sequences disclosed in FIG. pp32, pp32r1, and / or pp
The method of detection and / or discrimination between 32r2 is a preferred method. The circumstances in which discrimination between pp32 variants may be beneficial will be apparent to the skilled clinician in view of the present disclosure. The selection of diagnostic techniques of the technology provided by the present invention and suitable to achieve the desired benefits is routine for the skilled clinician.

[0059]

【Example】

Numerous embodiments are provided below to facilitate a more complete understanding of the present invention. However, the scope of the present invention is not limited to the particular embodiments disclosed in this example, which are for illustrative purposes only.

Example 1 Location of Cells Expressing pp32 pp32 mRNA can be detected under stringent conditions by in situ hybridization using a pp32 probe.

In situ hybridization Bases 1-298 of the pp32 cDNA sequence (GenBank HSU73477) were
It was subcloned into the ript vector. Digoxigenin-labeled antisense and sense RNA probes were prepared using a commercially available kit (Boehringer Mannheim). Vector DNA linearized with BamHl and Xhol was used as a template for the preparation of antisense and sense probes, respectively. 1 μg of template DNA, 2 U /
μl of either T3 or T7 RNA polymerase, 1 U / μl ribonuclease inhibitor, 1 mM each of ATP, CTP, GTP, 0.65 mM UTP, 0.35 mM digoxigenin-11-UTP, 4
In vitro transcription was performed at 37 ° C. for 2 hours in a final volume of 20 μl containing 0 mM Tris-HCl pH 8.0, 10 mM NaCl, 10 mM DTT, 6 mM MgCl ₂ , and 2 mM spermidine. 0.2
The reaction was stopped by adding 2 μl of M EDTA, pH 8.0, and then 2.2 μl of 4
Precipitation was carried out at -70 ° C. for 30 minutes using M LiCl and 75 μl of pre-cooled ethanol. Centrifuge the RNA to pellet, wash with 80% ethanol, dry gently,
Then, it was dissolved in 100 μl of DEPC-treated water. The yield of the labeled probe was determined by enzyme-linked immunoassay (enzyme linked i) using a commercially available kit (Boehringer Mannheim).
rrimunoassay). Non-radioactive in situ hybridizations were performed using antisense and sense pp32 RNA probes generated by in vitro transcription (US Pat.
5,726,018). FIG. 1A shows that normal prostate basal cells are positive, while clear differentiated gland cells are negative. Conversely, prostate adenocarcinoma is markedly positive, as shown on the left. The invention detects mRNA, not protein
Note that the signal is in the cytoplasm.

Pp32 shows a unique expression pattern in vivo (Chen, et al .; Malek, et al.
“Identification and preliminary characterization of two related prolife
ration-associated nuclear phosphoproteins. ”Journal of Biological Chemis
try, 265: 13400-13409, 1990; Walensky, et al., “A novel M (r) 32,000 nuclear
phosphoprotein is selectively expressed in cells competent for self-rene
wal, “Cancer Research 53: 4720-4726, 1993). In normal peripheral tissues, expression is restricted to a population of stem-like cells such as intestinal crypt epithelial cells and basal epithelium of the skin; Neurons in the cerebral cortex and Purkinje cells also express pp32. In normal prostate, basal cells express pp32, whereas in differentiated gland cells pp32 mRNA cannot be detected by in situ hybridization ( Figure 1A) .In comparison, strong in situ hybridization to the pp32 probe was found in almost all clinically relevant human prostate adenocarcinomas. Gleason score 4 and below in a study of 55 patients. 11 in cancer
% Of human prostate adenocarcinoma with a Gleason score of 5 and above
87% express mRNA that hybridizes strongly with the probe for pp32.

Immunohistochemistry Formalin-fixed, paraffin-embedded tissues were sectioned at 4 μM, deparaffinized, hydrated, 95% in 0.01 M citrate buffer, pH 6.0 for heat-induced antigen recovery.
C. for 20 minutes (Cattoretti, et al., “Antigen unmasking on formalinfixed, pa
raffin-embedded tissue sections, ”Journal of Pathology 171: 83-98,1993)
Then, it was incubated overnight at room temperature with a 1/20 dilution of the anti-pp32 antibody. Following washing, slides were serially diluted 1/100 in biotinylated porcine anti-rabbit IgG antibody (Dako)
, Streptavidin peroxidase (Dako) and diaminobenzidine. FIG. 1B shows a typical high grade human prostate cancer stained with affinity purified rabbit anti-pp32 polyclonal antibody (Gusev, et al., “Pp3
2 overexpression induces nuclear pleomorphism in rat prostatic carcinoma
cells, "Cell Proliferation 29: 643-653, 1996). The left panel shows a 250x representative field of view, and the rectangle shows the area where computer-generated details are shown in the right panel. Strong hybridization Tumors showed strong immunopositivity with the pp32 antibody, indicating that they expressed pp32 or immunologically relevant proteins (FIGS. 1A and 1B).

On the surface, the localization data shown in FIGS. 1A and 1B indicate that pp32 is expressed in both normal and neoplastic prostate epithelial cells, despite their ability to inhibit tumorigenic functions such as transformation. I showed you. The explanation for this apparently inconsistent expression is that prostate tumors do not express pp32 globally, but rather express variants of pp32 that promote, instead of inhibit, transformation.

Example 2 ESTs Corresponding to pp32 Some possible pp32 variants are identified in the NCI's Cancer Genome Anatomy Proj.
ect was identified from a prostate cancer expressed sequence tag library. Clone 588488 encodes a protein with 96% identity to APRIL, but without recovery and sequencing of the entire clone, it is impossible to tell whether the entire EST clone encodes a pp32-related sequence; It is not possible at this time to investigate the biological function. Nevertheless, the sequenced part is pp
It is clear that it encodes a protein with high homology to 32. This EST is not in the normal prostate database. Similar to the pp32 variant removed from prostate cancer, the creation of this molecule by mutation would require a complex mechanism.

[0066] The pp32-related gene is also present in other organisms. The presence of the pp32 gene family in rodents will not be inconsistent with the existence of comparable sized families in humans. Mouse pp32 (GenBank U73478) has 89% amino acid identity with pp32,
Less identity with pp32r1 and APRIL (mouse cerebellar leucine-rich acidic nucleoprotein has one amino acid substitution compared to mouse pp32). We further identified a mouse EST, GenBank AA066733, with 85% identity and the highest identity to the APRIL protein, over 148 amino acids of the predicted open reading frame. Some other mouse ESTs, AA212094 and W82526, are closely related to the pp32 family, but are not significantly more related to either pp32, pp32r1 or APRIL. The human homolog of the gene is
It could be expected to code the fourth member. We identified ESTs predicted to encode pp32-related proteins in C. elegans, schistosomes, zebrafish, and Drosophila (data not shown). However, these sequences cannot represent the entire range of the pp32 gene family in these organisms, and thus do not give information about the appropriate size of the mammalian pp32 gene family.

Example 3 Gene Structure Encoding a pp32 Family Relative A human genomic library integrated into a bacteriophage was transformed into a human pp32 cDN
Screening with the probe generated from A yielded a new sequence containing an open reading frame encoding a protein homologous to pp32.

Screening of human genomic library integrated into bacteriophage against pp32 cDNA The genomic library from human placenta integrated into Lambda Fix II vector was
coli strain XL-1 Blue MRA (Stratagene # 946206). Screening for bacteriophage clones containing DNA inserts homologous to the pp32 cDNA followed routine procedures (Sambrook, et al.). Briefly, P-32 labeled pp32 c nitrocellulose filters placed on bacteriophage plaques
Hybridized with a probe for DNA. Probes were prepared by the random primer method (Stratagene # 300385) using pp32 cDNA as a template (Chen, et al., Molec. Biol. Cell, 7: 2045-2056, 1996). Reactive bacteriophages were plugged and the bacteriophages were eluted, re-expressed, and rescreened to purity using the pp32 cDNA probe. Bacteriophage DNA was prepared by the plate lysate method (Sambrook, et al.).

Identification of restriction fragments in bacteriophage DNA containing sequences homologous to pp32 cDNA DNA from a bacteriophage clone containing the pp32 cDNA sequence was digested with HindIII. Using routine methods, the restriction fragments were separated by agarose gel electrophoresis,
Transferred to a positively charged nylon filter in alkaline buffer and hybridized with selective probes at the 5 'and 3' ends of the pp32 cDNA (Sambrook, et al.). The 5 'and 3' probes were prepared as described above except that the product of the polymerase chain reaction (PCR) was used as a template for the labeling reaction (Saiki, et al., Science, 239: 487-491. , 1988). One PCR product
It was a portion of a 249 base pair pp32 cDNA containing nucleotides 32 to 279. It is p
The results were obtained using a p32 cDNA template and the primers 5′-TATGCTAGCGGGTTCGGGGTTTATTG-3 ′ and 5′-GATTCTAGATGGTAAGTTTGCGATTGAGG-3 ′ (primer set A). The other product was a portion of a 263 base pair pp32 cDNA containing nucleotides 677 to 938. It was the result of a reaction using the pp32 cDNA template and the primers 5'-GAATCTAGAA GGAGGAGGAA GGTGAAGAG-3 'and 5'-CTATCTAGAT TCAGGGGGCAGGATTAGAG-3' (primer set B). The PCR reaction included 35 cycles of 1 cycle of denaturation at 95 ° C for 1 minute, annealing of primers at 50 ° C for 1 minute, and extension at 72 ° C for 1 minute (cycling program A). A 4.7 kb HindIII restriction fragment that hybridizes with the 5 ′ probe but not the 3 ′ probe, and hybridizes with the 3 ′ probe but not the 5 ′ probe, 0.9 kb HindI
The II restriction fragment was subcloned into pBluescript (Gibco) by routine methods (Sambrook, et al.). The nucleotide sequences of both strands of the purified plasmid DNA, including the insert, were determined by an automated method (DNA Analysis Facility, Johns
Hopkins University School of Medicine).

Completion of Sequencing by Direct Sequencing of PCR Products The sequence of the 4.7 and 0.9 kb Hind III restriction fragments and the pp32 cDNA were aligned (alignment
t) between the 3 'end of the 4.7 kb fragment and the 5' region of the pp32 cDNA, and the 5 'end of the 0.9 kb fragment.
It showed about 90% homology with the 3 'region of pp32 cDNA. There was a 199 base pair gap between the ends of the restriction fragment that did not match the pp32 cDNA. Primers were designed to specifically anneal to the relevant pp32 sequence on either side of the sequence gap. The primer sequences were 5'-GAGGTTTATT GATTGAATTC GGCT-3 'and 5'-CCCCAGTACA CTTTTCCCCCCTCTCA-3' (primer set C). The polymerase chain reaction was then performed with cycling program A using primer set C and purified bacteriophage DNA as template. Ethidium bromide staining of agarose gel showed 943 bp product, extracted from fragments excised from low melting point agarose (NuSieve) electrophoresis gel and sequenced in an automated manner as described above. .

A 5,787 base sequence was obtained from a bacteriophage clone of a human placenta genomic library containing portions homologous to the pp32 cDNA (FIG. 2). This sequence was deposited at Genbank under accession number U71084, Locus HSU71084. The sequence is nucleotide 4,
It has an open reading frame ranging from 453 to 5,154. Analysis of the nucleotide sequence upstream of the open reading frame revealed more than 20 consensus sequences for the active steroid hormone receptor (Table 1).

Sequence analysis of the open reading frame showed 94% sequence homology with pp32 (FIG. 3). Alignment of the open reading frame sequence with the pp32 cDNA revealed 33 single base differences and a clustered difference of 2 and 7 bases. There were two internal deletions of 3 and 9 bases. The open reading frame encoded a polypeptide containing 234 amino acid residues with 88% homology at the protein level with pp32 (FIG. 4). By aligning the translated sequence with the pp32 amino acid sequence, differences in 18 single amino acid residues, 3 differences in a 2-residue cluster, and 1 difference in a 4-residue cluster It was revealed. There were two internal deletions of 1 and 3 residues and a terminal deletion of 11 residues. The translated sequence contained 26 fewer, 69 acidic residues than pp32.

Example 4 Chromosome Mapping of pp32rl NIGMS Monochromosomal Panel 2 (Drwinga, et al., “NIGMS human / ro
dent somatic cell hybrid mapping panels 1 and 2, ”Genomics 16: 311314,19
The pp32r1 gene is located on chromosome 4 as determined by PCR of 93) followed by sequencing of the PCR product. Interestingly, 43 of the sequence 5 'to the starting ATG
The entire sequence of pp32r1 including 64 nucleotides is a non-redundant Gen
Included more than 400 matches in the blastn search of the Bank database. These matches show repeats in the opposite orientation, about 278
And two short regions of 252 base pairs (nucleotides 674-952 and 2542-2794). This repeat is significantly associated with elements on many chromosomes.

The human pp32 gene was cloned into chromosome 15q22 by fluorescence in situ hybridization.
Mapped to .3-q23 (Fink, et al.). pp32 (Hs.76689; HLA-DR related protein 1)
The single gene entry to lists the 93 ESTs corresponding to this gene, 12 of which contain a mapped sequence-tagged site (STS). Like many pp32 ESTs analyzed by electrical PCR, all of these STS sites
(Http://www.ncbi.nlm.nih.gov). The APRIL protein has also been mapped to chromosome 15q25 (Mencinger et al .; GenBank Y07969).

Example 5 Sequence Analysis of pp32r2 A pp32-related sequence (designated pp32r2) was described in Example 2 to isolate the unique intronless pp32-related gene pp32r1 found on chromosome 4. In a manner similar to that identified on chromosome 12. It was initially thought that the sequence on chromosome 12 encodes a truncated protein and may represent a pseudogene; however, its interpretation was reviewed in light of this finding. The sequence is pp32r2
And registered in Genbank as locus AF008216; the sequence of pp32r2 is
Shown in BESTFIT analysis (Genetics Computer Group, Inc., Wisconsin Package, v
ersion 9.1, Madison, WI, 1997) shows that pp32r2 has FT1.11, FT2.4, and T1 and 99
It is .5% identical and shows 4 nucleotide differences over an 845 nucleotide overlap of the sequence; this level of diversity is consistent with the polymorphism. BESTFIT
Analysis shows that PP32R1 is 99.6% identical to FT3.3 and 99.4% identical to FT2.2,
They show a difference of 4 and 5 nucleotides, respectively (see Figure 7, bottom).

Example 6 Sequence Comparison of Various Clones A human placenta genomic library integrated into a Lambda Fix II vector (Stratagene # 946206) was screened with a P-32 labeled probe against pp32 cDNA, and an approximately 23 kb clone was cloned. Obtained. The 4.7 kb and 0.9 kb HindIII restriction fragments of this clone were hybridized with a probe for pp32 cDNA. The 4.7 kb clone was aligned with the 5 'portion of the pp32 cDNA sequence, and the 0.9 kb fragment was aligned with the 3' end. A small break of 0.2 kb was sequenced from the bridging PCR product. No intron was identified.

The whole human embryo strain FSH173WE and the prostate cancer cell lines PC-3 and LNCaP (American Type
Culture Collection) was grown under recommended tissue culture conditions. Poly A + RNA was prepared by oligo dT adsorption (MicroFasTrack, Invitrogen), and reverse transcriptase and random hexamers (GeneAmp RNA PCR Kit, Perkin Elme)
Used as a template to generate cDNA by reaction using r). The cDNA sequence encoding the open reading frame was amplified by nested PCR using primers specific for the genomic sequence, including the pp32 sequence. A final product of 298 base pairs was seen by bromide bromide staining of the agarose electrophoresis gel.

In most cases, DU-145 cells and transcripts from a number of patients were obtained from the above samples using a method similar to that described in Example 3, except that no nested primers were required. Were sequenced for comparison with the transcript of The results are shown in Table 2. A summary of the degree of identity between the various transcripts is provided in Table 3.

Example 7 Sequence Diversity of Individual Isolates from Different Cell Lines and Tumor Tissues An explanation for the apparently inconsistent expression of pp32 in cancer is that prostate tumors do not express pp32 globally, but rather To express a pp32 variant that promotes transformation instead of inhibiting it.

RT-PCR and CFLP With the Titan One-Tube RT-PCR kit (Boehringer), bases 32 to 52 of HSU73477 were used as a forward primer, and 91% of the same sequence was used as a reverse primer.
Sequences were reverse transcribed and amplified using 9-938. Reverse transcription is performed at 50 ° C for 45 minutes, then at 94 ° C
The PCR was followed by a PTC 100 thermocycler (MJ Research
), 45 cycles were performed, each cycle consisting of 92 ° C. for 45 seconds, 55 ° C. for 45 seconds, and 68 ° C. for 1 minute, and a final extension at 68 ° C. for 10 minutes. Template RNA was isolated from cell lines or frozen tumor specimens using RNA sol B (Tel-Test) according to the manufacturer's instructions and then digested with RNAse-free DNAse I (Boehringer). pCMV32 was used as a positive control without reverse transcription. According to the manufacturer's specifications (Life Technologies), 5
Cleavage assays were performed by digestion with 0.2 mM MnCl ₂ at 5 ° C. for 10 minutes and electrophoresed on a 6% denaturing polyacrylamide sequencing gel.

At the level of RT-PCR, amplification products ranging from 889 to 909 bp (FIG. 6A) were obtained from paired normal prostate and prostate adenocarcinoma from three patients. The reaction is pp32,
And, consensus primers were used that could amplify the full length of the coding sequence from two closely related intronless genomic sequences, pp32r1 and pp32r2. The only noticeable difference was the lower amplicon yield from normal tissue compared to cancer. Four human prostate adenocarcinoma cell lines DU-145, LNCaP, PC-3, and TSUPR-1 also obtained similar products.

FIG. 6A shows DNA from human prostate and prostate cancer cell lines amplified by RT-PCR. Lane a is the undigested control, the band migrating substantially slower than the digestion product; samples from all other lanes were digested by cleavage as described.
Lanes: 1 kb ladder (Life Technologies), A; pCMV32, B; DU-145, C; LNCa
P, D; PC-3, E; TSUPr-1, F; FT-1, G; FN-1, H, which are representative samples without reverse transcription
FT-1, I; FN-2, J; FT-2, K; FN-3, L; FT-3, M; negative control without template. FN indicates frozen benign prostate and numbers indicate patients; FT indicates frozen prostate adenocarcinoma and numbers indicate patients. The numbers on the left side of the figure indicate the 1 kb DNA ladder (Li
fe Technologies) in kb.

When the RT-PCR product was subcloned and analyzed by fragment length polymorphism analysis (CFLP) and sequence analysis, qualitative differences between normal and neoplastic tissue began to be revealed Was. FIG.6B shows the fragment lengths of cDNAs amplified and cloned by RT-PCR from human prostate adenocarcinoma, adjacent normal prostate, and human prostate adenocarcinoma cell lines using primers derived from the normal pp32 cDNA sequence. 3 shows polymorphism analysis. Lane is DU
-145, showing clones from RT-PCR from LNCaP, PC-3 and TSUPr1 cell lines, from frozen prostate cancer (FT), and from frozen normal prostate (FN) respectively: a,
Undigested normal pp32 cDNA; b, normal pp32 cDNA; c, DU-145-1; d, DU-145-3; e, DU-1
45-5; f, LNCaP-3; g, PC3-3; h, PC3-8; I, TSUPr1-1; j, TSUPr1-3; k, TSUPr
1-6; l, FT1.11; m, FT1.7; n, FT2.2; o, FT2.4; p, FT3.18; q, FT3.3; r, FN
3.17; s, FN2.1. LNCaP normally expresses pp32. Band shifts correspond to sequence differences. All clones of RT-PCR products from normal prostate tissue are cloned
A normal CFLP pattern is shown that exactly matches that obtained from the pp32 cDNA template (GenBank HSU73477, FIG. 6B). Prostate adenocarcinoma has 4 different Cs in similar analysis
Get FLP patterns, three of which are unique and one is normal pp32
Similar to the pattern. Examination of DU-145, PC-3, and TSUPR-1 cell lines yielded roughly the same results, but LnCaP gave only normal pp32 CFLP patterns. Further analysis at the sequence level confirmed that normal prostate and LnCaP contained only normal pp32 transcript.

Prostate adenocarcinoma and transcripts from most cell lines are closely related
The variants of pp32 are shown and are outlined in Table 1. These transcripts are normal pp32 cDNA and 92.4
% To 95.9% different nucleotide identities (Genetics Computer Group, I
nc., Wisconsin Package, version 9.1, Madison, WI, 1997). Of the 16 mutant transcripts obtained, 15 have an open reading frame encoding a protein with identity ranging from 89.3% to 99.6% with normal pp32. The table summarizes data obtained from mutant pp32 transcripts obtained from human prostate adenocarcinoma and prostate cancer cell lines. Sequences that fall into closely related groups are indicated by group letters (A, B, C); U indicates sequences that do not clearly belong to the group and cannot be assigned. The origin of each sequence is: FT, frozen tumor,
Subsequently, the number of patients, decimal point, and clone number; D, DU-145, followed by clone numbers (similar to all cell line sequences); P, PC3; and T, TSUPrl. Nucleotide identity, nucleotide sequence gap, sequence comparison, and protein identity
Determined from BESTFIT alignment using pp32 cDNA and protein sequences. The effects on transformation were: stimulation, more cell foci were obtained when transfected with ras + myc than ras + myc + vector control; inactive, ras
Cell foci equivalent to + myc + vector control are obtained; and suppression, ras + myc +
It states that fewer foci were obtained than the vector control.

The predicted protein sequences were divided into three separate groups: [1] N-terminal 131 of pp32
One of said sequences, roughly equivalent to pp32r2, with truncated sequences spanning amino acids, was obtained from two of three patients and from the TSUPR-1 cell line; [2] a different p
p32-related genes, homologous sequences closer to pp32r1, and [3] foreign pp32-related sequences. Tumors from two of the three patients analyzed contained no detectable normal pp32 transcript. Two of the twelve cloned transcripts from the third patient's tumor were normal by CFLP pattern, and one clone also confirmed sequence normality. Two clones from the cell line were normal by CFLP screening, but were later shown to display mutated sequences.

[0086] Contrary to the prostate of both sexes, where sequence analysis yielded only pp32, adjacent prostate cancers expressed little or no pp32. Instead, four pp32-related transcripts with different sequences, encoding open reading frames,
92.4% nucleotide identity to normal pp32 cDNA from adjacent prostate cancer
To 95.9%. Of these, two transcripts, corresponding to the products of the previously identified pp32-related genes, pp32r1 and pp32r2, were obtained many times from patient samples and were therefore the focus of this study.

FIGS. 7A and 7B show the nucleotides of all transcripts and a number of paired alignments of the predicted protein sequence (Smith, et al., “Identification of com.
mon molecular subsequences, ”J. Mol. Biol., 147: 195-197 1981.
Edited using Pileup and Pretty programs (Smith, et al.). Differences from the consensus sequence are shown as indicated; matches with the consensus sequence are shown as blanks. Normal human pp32 is designated as hpp32. Sequences from TSUPr1, PC3 and DU-145 cell lines are as shown. The FT designation shows sequences from frozen human prostate adenocarcinoma. Normal pp
Only 32 sequences, hpp32, were obtained from normal prostate adjacent to tumor tissue. Figure 8A shows pp3
2 shows the alignment of the amplicon nucleotide sequence with the amplicon predicted from 2 and pp32r1; FIG. 8B shows the alignment of the predicted protein sequence. One sequence (FT 1.11) obtained three times independently from two other patients and the TSUPR-1 cell line is shown only once in the diagram. Pileup and pairwise alignments explain several important points: [1] The sequence is highly conserved, both at the nucleotide and predicted amino acid levels;
[2] Sequence differences are distributed over the entire length of the sequence without obvious hot spots; [3] There is no apparent clustering or partitioning of sequence differences; and [4]
Various sequences have been applied to the groups described above. These points are detailed in FIGS. 8A and 8B.

The same pp32r1 sequence obtained from the two patients differed by 4 nucleotides from the expected product of the pp32r1 genomic sequence. The sequences of pp32r2 obtained from the two patients were also identical and differed by 3 nucleotides from the pp32r2 genomic sequence. Both sets of differences are considered consistent with polymorphic diversity. As shown in the multiple pairing alignments of the expected protein sequences in FIG. 7, pp32, pp32r1, and pp32
32r2 shows sequence changes over its entire length. Simple methods of somatic mutation or alternative splicing could not explain these results. Instead,
According to the correspondence between the previously identified genes on chromosomes 4 and 12 and the mutated sequence,
The data is consistent with selective gene expression.

Example 8 Diagnostic Methods to Identify Family Members The three members of the pp32 family expressed in human prostate cancer are pp32, pp32r1
, Pp32r2. pp32 transforms in vitro and in vivo in model systems
Pp32r1 and pp32r2 inhibit pre-transformation (pr
o-transforming) and tumorigenic. Using a protocol similar to that described in Example 7, it is possible to determine which of the three members is expressed in the tissue sample.

Analysis from Fresh Frozen Human Tissues and Cell Lines Extracting total RNA from fresh frozen human tissues or human cancer cell lines, reverse transcription and polymerase chain reaction amplification were performed on all three genes. Using a set of primers that can amplify the entire coding region of the cDNA. A suitable set of primers is: Upstream: 5'GGGTTCGGGG TTTATTG3'-this is the pp32 cDNA sequence (Genbank U73477)
corresponds to bp 32 to bp 48; downstream: 5'CTCTAATCCT GCCCCCTGAA3'-this is the pp32 cDNA sequence (Genbank U73477
) Corresponding to bp 919 to bp 938. The sizes of the amplicons observed using this primer set are pp32-907 bp, pp32r1-889 bp and pp32r2-900 bp. The three cDNA sequences are distinguished from each other by restriction enzyme digestion with the following enzymes-EcoR I, Hind III and Xho I. The resulting digest is run on a 2.5% agarose gel and the three different cDNAs are clearly distinguished. The table below tabulates the observed band sizes. Bold numbers indicate useful band sizes for identification of the three cDNAs.

[0091]

[Table 1]

Analysis from Formalin-Fixed and Paraffin-Embedded Tissues A similar approach follows for the identification of pp32, pp32r1, and pp32r2 transcripts from formalin-fixed and paraffin-embedded tissues. Total RNA is extracted and reverse transcription and PCR amplification is performed using a set of primers capable of amplifying 200 bp in length from all three cDNAs. A suitable set of primers is: bp 394 to bp 414 of the upstream primer-pp32 cDNA sequence (Genbank U73477) bp 609 to bp 629 of the downstream primer-pp32 cDNA sequence (Genbank U73477) The three cDNA sequences are: -Differentiated from each other by restriction digestion with Hind III, Xho I and BseR I. The resulting digest is run on a 3% agarose gel and the three different cDNAs are clearly distinguished. The table below tabulates the observed band sizes. Bold numbers indicate useful band sizes for identification of the three cDNAs.

[0093]

[Table 2]

Example 9 pp32r1 Increases Oncogene-mediated Transformation of Rat Embryonic Fibroblasts pp32r1 is subcloned downstream of the CMV promoter in a eukaryotic expression vector and ras + myc is expressed in rat embryonic fibroblasts. Was analyzed for its effect on the formation of cell foci transformed via E. coli. The genomic sequence containing the entire coding region of pp32r1 was amplified by PCR and subcloned into the eukaryotic TA cloning and expression vector containing the CMV promoter, the pCR3.1 vector (Invitrogen). 5 micrograms of pEJ-ras and / or 10 micrograms of pMLV-c-myc
Each T that received only pCMV32, pp32r1 incorporated into PCR3.1, or PCR3.1
Using 75 flasks, assay as described (Chen et al., Mol Biol Cell. 7: 2045
-56, 1996). After 14 days, transformed colonies were counted. FIG. 8 shows the results. Data consisted of two separate experiments performed in duplicate and one performed in triplicate7
The average of the number of repetitions is shown. Error bars indicate standard error of the mean. In contrast to pp32, which consistently suppresses cell foci formation induced by the combination of ras + myc and other oncogenes, pp32r1 has a statistically significant p = 0.04 by unpaired t-test Stimulation of cell foci formation was caused.

Example 10 Effect of Transcripts from Various Cell Lines on the Rat Fibroblast Transformation Assay As described above, expression constructs prepared from PC-3 and DU-145 cells were used herein. Chen, et al. (Mol Biol Cell., 7: 2045-56, 1996, incorporated by reference.
) Was tested in a rat embryo fibroblast transformation assay as described in). The results are shown in FIG. Unlike normal pp32, which suppressed transformation, transcripts from two cell lines stimulated ras + myc induction of transformed rat embryo fibroblast cell foci. The figure shows the mean +/- standard deviation, except for DU-145, which indicates one decision.

Example 11 Transformation Activity of Various Isolates from Patient Tumors Mutant transcripts isolated from prostate cancer patients differ significantly in sequence from pp32. The isolated transcript was found to stimulate transformation.

Transformation Assay Rat embryonic fibroblasts were transfected with the indicated constructs as described (Chen, et al.) And transformed cell foci were enumerated. In each experiment, approximately 1 × 10 ⁶ cells were seeded per T75 flask and incubated for 2-3 days before transfection to reach approximately 40% confluence. To each flask of primary rat embryo fibroblasts, the following amounts of the plasmid indicated in each experiment were added: pEJ-ras, 5 μg; and pMLV-c-myc, pCMV32, pCMVneo, or pCR3.1 (Invitrogen). Incorporated mutant pp32 construct, 10 μg. Plasmids were prepared in 2 volumes of Lipofectin (2 μl lipofectin per μg DNA), then gently mixed in 1.5 ml OPTMEM up and down in sterile 15 ml polystyrene tubes and incubated at room temperature for more than 15 minutes. For experiments using more than one flask, the mixture of all reagents was increased in proportion to the number of flasks required for each transfection. Wash cells once with OPTIMEM (Gibco-BRL)
Thereafter, 6 ml of OPTIMEM and 1.5 ml of DNA / Lipofectin mixture were given. After overnight incubation, cells were grown in standard medium and fed twice a week with fresh medium. Cell foci were counted 14 days after transfection. FIG. 10 summarizes four separate experiments. Each data point represents the results from each flask, expressed as a percentage of the foci obtained from a concurrent ras + myc + vector control.

FIG. 10 shows that mutated transcripts derived from prostate cancer cell lines and human prostate adenocarcinoma were co-transfected with ras and myc, ras and myc when transfected with empty vector. This generally produces an increase in the number of transformed cell foci as compared to the number of cell foci obtained. From DU-145 (D3) and 3
Mutant pp32 transcripts from two prostate cancers (FT 1.7, FT 2.2, and FT 3.18)
And an increase in the number of transformed foci over that produced by only myc and the empty vector. This is a clear difference from normal pp32, which consistently suppresses transformation. These activities are also outlined in Table 1.

Example 12 Effect of pp32 Mutants on Tumorigenicity In Vivo Experiments testing the effect of transfection of NIH3T3 cells on tumorigenicity in vivo were performed using rat embryo fibroblasts in vitro. Consistent with the results in NIH3T3 cells were stably transfected with lipofectiin using the pp32 species shown in Table 6A in a PCR3.1-single CMV driven mammalian expression vector (Invitrogen). All G418 resistant clones used in these experiments were shown by genomic PCR to contain the indicated pp32 species. For the analysis of tumorigenicity, 5 × 10 ⁶ cells in 100 microliters of Dulbecco's modified Eagle's medium without phenol red were added to thymus-free, female nude mice over 6 weeks of age in an inbred background. (Harlan) was injected into the flank. For logical reasons, the inoculations of the various groups were shifted by more than 7 days. Mice in each group were euthanized exactly 7 weeks after inoculation. If the mouse has a tumor, the tumor is excised, measured and weighed, and Table 6A reports the average tumor weight in mice injected with cells with the various vectors. One tumor from each group was examined histologically. All tumors were fibroblast sarcomas and there were no notable inflammations. Data obtained for NIH3T3 cells show NIH3T3 cells stably transfected with various pp32 variants, P3, P8, FT1.
7 shows that FT2.2 and FT2.4 form tumors when inoculated into nude mice. In contrast, NIH3T3 stably transfected to express human pp32
The cells did not form tumors in vivo, even when transfected with ras. A line of NIH3T3 cells stably transfected with an expression construct encoding pp32 or pp32-antisense was also established. Basal expression of pp32 is essential for contact inhibition and maintenance of serum-dependent cell proliferation; antisense depletion of endogenous pp32 synthesis allowed cells to grow normally after serum removal. Constitutive overexpression of pp32 effectively suppresses ras-mediated transformation of NIH3T3 cells in vitro and tumorigenicity in vivo. In contrast, antisense depletion of endogenous pp32 dramatically increased the number and size of ras-transformed foci; obtained in vivo from ras-transformed antisense pp32 cells Tumors were approximately 50 times as large as tumors obtained from control cells transformed with ras.

The oncogenic switch expressing pp32 family members is the same as the switch in the pp32 phenotype of prostate cancer. Example 11 shows that when pp32r1 and pp32r2 are expressed at a high frequency compared to the number of cell foci obtained when ras and myc are transfected with an empty vector, co-transfection with ras and myc Shown are experiments showing that transformed foci formation cannot be inhibited or sometimes stimulated. This means that normal pp32 consistently suppresses transformation,
There is a clear contrast. Similarly, Example 12 shows pp32 vs. non-tumorigenic pp32.
It shows that both 32r1 and pp32r2 are tumorigenic when stably transfected into NIH3T3.

Other aspects, advantages, and modifications will be apparent to those skilled in the art to which the invention pertains, but for clarity of understanding, the foregoing invention, as well as certain aspects, will be described in some detail by way of illustration and example. The details have been described. The foregoing description and examples are intended to be illustrative, but do not limit the scope of the invention. Modifications of the above-described methods for carrying out the invention which are obvious to those skilled in medicine, immunology, hybridoma technology, pharmacology, pathology, and / or related fields are within the scope of the invention. And is limited only by the appended claims.

All publications and patent applications mentioned herein are indicated at the level of those skilled in the art to which this invention belongs. All publications and patent applications are herein incorporated by reference to the same extent as if each publication or patent application was specifically and individually indicated to be incorporated by reference. You.

[0103]

[Table 3]

[0104]

[0105]

[0106]

[0107]

[0108]

[0109]

[0110]

[0111]

[Table 4]

[0112]

[0113]

[Table 5]

[0114]

[Table 6]

[0115]

[Table 7]

[0116]

[Table 8]

[0117]

[Table 9]

¹ FT1.7, clone 2 and vector control, clone 3, were tested on the contralateral sides of one group of animals. ² D3 clone 5 was tested on both contralateral sides of a group of animals co-injected with NIH3T3 cells transfected with the pp32r1 clone (data not shown). D3
Clone 6 was tested on both contralateral sides of a group of animals co-injected with a second clone of NIH3T3 cells transfected with the pp32r1 clone (data not shown). ³ P3, clone 14, and vector control, clone 2, were tested on both sides of one contralateral animal group. ⁴ P8, clone 1 and pp32, clone 6 were tested on both contralateral sides of one group of animals. ⁵ P8, clone 4 and pp32, clone 5 were tested on both contralateral sides of one group of animals. ⁶ One tumor weighing 0.5 gm in this group was detected only after post-mortem dissection.

[Brief description of the drawings]

FIG. 1A shows in situ pp32-related mRNA in benign prostate and prostate cancer tissue sections.
The detection by tu hybridization is shown. FIG. 1B shows immunohistochemical staining of prostate cancer sections using anti-pp32 antibody.

FIG. 2 shows the genomic sequence of mutant pp32r1 isolated from human placenta.

FIG. 3 provides a base-to-base comparison of the sequences of pp32r1 (top) and normal human pp32 (bottom). The numbering system for pp32r1 is consistent with FIG. 1, and the numbering system for normal pp32 is from Chen et al. Nucleotide base differences are underlined in the pp32r1 sequence. Sequences within the normal pp32 sequence that are missing in pp32r1 are indicated by dashes. The open reading frame of pp32r1 is indicated by overlining.

FIG. 4 shows the arrangement (alignme) of the amino acid sequence of pp32r1 (upper) and normal human pp32 (lower).
nt). Residue changes are underlined in the pp32r1 sequence. Pp32r1 compared to normal pp32
Amino acids missing in the sequence are indicated by dashes.

FIG. 5 shows the genomic sequence of mutant pp32r2.

FIG. 6A shows RT of pp32 and pp32 mutants from human prostate cancer and prostate cancer cell lines.
-PCR amplification is shown. FIG. 6B shows that mutant pp32 transcripts are detected in human prostate cancer by cleavage fragment length polymorphism analysis of pp32.

FIG. 7 shows nucleic acid derived from human prostate adenocarcinoma and prostate adenocarcinoma cell line using pp32 (A)
And the arrangement of the amino acid (B) sequence.

[Fig. 8] Fig. 8 shows that ras + myc is transformed focus formation.
7 is a bar graph showing that was induced. Co-transfection with the pp32 expression vector reduces transformation, while co-transfection with the pp32r1 expression vector stimulates transformation.

FIG. 9 is a bar graph showing that pp32r1 stimulation of ras + myc induced transformed cell foci formation. Co-transfection with the pp32 expression vector reduces transformation, whereas co-transfection of the pp32r1 sequence from a prostate cancer cell line with the expression vector stimulates transformation.

FIG. 10 is a graph of the results of a transformation assay on cells transfected with the mutant pp32 species, showing that transformation is stimulated with various efficiencies.

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat ゛ (Reference) G01N 33/15 G01N 33/15 Z 33/50 33/50 Z // C12N 15/09 ZNA C12N 15/00 ZNAA (81) Designated countries EP (AT, BE, CH, CY, DE, DK, ES, FI, FR, GB, GR, IE, IT, LU, MC, NL, PT, SE), OA (BF, BJ, CF, CG, CI, CM, GA, GN, GW, ML, MR, NE, SN, TD, TG), AP (GH, GM, KE, LS, MW, SD, SL, SZ, TZ, UG, ZW), EA (AM, AZ, BY, KG, KZ, MD, RU, TJ, TM), AE, AL, AM, AT, AU, AZ, BA, BB, BG, BR, Y, CA, CH, CN, CR, CU, CZ, DE, DK, DM, EE, ES, FI, GB, GD, GE, GH, GM, HR, HU, ID, IL, IN, IS, JP , KE, KG, KP, KR, KZ, LC, LK, LR, LS, LT, LU, LV, MA, MD, MG, MK, MN, MW, MX, NO, NZ, PL, PT, RO, RU, SD, SE, SG, SI, SK, SL, TJ, TM, TR, TT, TZ, UA, UG, US, UZ, VN, YU, ZA, ZWF terms (reference) 2G045 AA35 BB24 BB51 CB01 FB02 FB03 FB05 4B024 AA01 AA12 BA80 CA04 CA09 CA12 EA03 HA14 HA17 4B063 QA01 QA17 QA19 QQ08 QQ33 QR13 QR77 4C084 AA01 AA02 AA03 AA13 AA17 BA44 CA53 DC32 ZB262 ZC202 4C086 AA01 AA01A02 ZA14

Claims (11)

[Claims] 1. A method for treating a malignant cell, comprising restoring pp32 function to a malignant cell showing subnormal expression of normal pp32 or overexpression of a pp32 mutant. 2. A method for treating malignant cells, comprising inducing an increase in the expression of normal pp32 in the malignant cells of claim 1, which shows subnormal expression of normal pp32 or overexpression of a pp32 mutant. 3. The method of claim 2, wherein increased normal pp32 expression is induced by transfection of DNA encoding normal pp32. 4. The method of claim 2, wherein the increase in normal pp32 expression is induced by administering an inducer of normal pp32 expression. 5. An increase in pp32 expression in the presence of the compound indicates that the compound is an inducer, and therefore measures pp32 expression by cells cultured in the presence and absence of the compound. A method of screening for determining whether said compound is an inducer of pp32 expression, comprising: 6. Since the phosphatase is inhibited by the incubation with pp32, the method comprises inhibiting protein phosphatase in malignant cells showing subnormal expression of normal pp32 or overexpression of pp32 mutant. 2. The method of claim 1, wherein the cell is treated. 7. The method according to claim 7, wherein the phosphatase is a nucleoprotein phosphatase.
7. The method according to claim 6, wherein malignant cells showing subnormal expression of normal pp32 or overexpression of pp32 mutant are treated. 8. A method for treating a malignant cell, which comprises inhibiting the nucleoprotein phosphatase that is a member of the protein phosphatase 2A group, in a malignant cell showing subnormal expression of normal pp32 or overexpression of a pp32 mutant. Claim
7 ways. 9. Expression of pp32 in cells cultured in the presence and absence of a compound inhibits protein phosphatase activity, and inhibition of protein phosphatase by said compound causes said compound to inhibit pp32 function in said cell. Measuring the protein phosphatase activity in the cell, since it indicates that it is inducing. A method of screening for determining whether the compound is an inducer of pp32 function. 10. The expression of pp32 in cells cultured in the presence and absence of a compound inhibits nucleoprotein phosphatase activity, and the inhibition of nucleoprotein phosphatase by said compound results in the compound being pp32 in the cell.
Measuring nucleoprotein phosphatase activity in the cells, as indicating that it induces function, comprising: determining whether the compound is an inducer of pp32 function.
Screening method. 11. The expression of pp32 in cells cultured in the presence and absence of a compound inhibits the activity of nucleoprotein phosphatase, a member of the protein phosphatase 2A group, and the inhibition of nucleoprotein phosphatase by said compound Determining that the compound is an inducer of pp32 function, comprising: measuring nucleoprotein phosphatase activity in the cell, since the compound indicates that the compound induces pp32 function in the cell. Claim 10 to do
Screening method.